Glucagon antagonists

ABSTRACT

The present invention concerns therapeutic agents that antagonize the activity of glucagon. In accordance with the present invention, the compounds of the invention comprise: 
     a. a glucagon antagonist domain, preferably the amino acid sequence of SEQ ID NO: 7, or sequences derived therefrom by phage display, RNA-peptide screening, or the other techniques; and 
     b. a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred; 
     wherein the vehicle is covalently attached to the glucagon antagonist domain. The vehicle and the glucagon antagonist domain may be linked through the N- or C-terminus of the glucagon antagonist domain. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain.

This application claims the benefit of U.S. Provisional application Serial No. 60/201,436, filed May 3, 2000, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

A need exists for recombinant or modified therapeutic agents having glucagon antagonist activity.

Recombinant and modified proteins are an emerging class of therapeutic agents. Useful modifications of protein therapeutic agents include combination with the “Fc” domain of an antibody and linkage to polymers such as polyethylene glycol (PEG) and dextran. Such modifications are discussed in detail in a patent application entitled, “Modified Peptides as Therapeutic Agents,” U.S. Ser. No. 09/428,082, PCT appl. No. WO 99/25044, which is hereby incorporated by reference in its entirety.

A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).

Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference in its entirety). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.

Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion (Smith et al. (1993), Mol. Pharmacol. 43: 741-8) to search for peptides with favorable therapeutic properties. Hereinafter, this and related methods are referred to as “yeast-based screening.”

Still other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ peptides linked to RNA; for example, PROfusion technology, Phylos, Inc. See, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.

In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as “rational design.” In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an “alanine walk” or an “alanine scan.” When two residues (contiguous or spaced apart) are replaced, it is referred to as a “double alanine walk.” The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.

Structural analysis of protein—protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. Hereinafter, these and related methods are referred to as “protein structural analysis.” These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.

Conceptually, one may discover peptide mimetics or antagonists of any protein using phage display, RNA-peptide screening, yeast-based screening, rational design, and the other methods mentioned above.

SUMMARY OF THE INVENTION

The present invention concerns therapeutic agents that have glucagon antagonist activity with advantageous pharmaceutical characteristics (e.g., half-life). In accordance with the present invention, such compounds comprise:

a) a glucagon antagonist domain, preferably having very little or no glucagon agonist activity, or sequences derived therefrom by rational design, yeast-based screening phage display, RNA-peptide screening, or the other techniques mentioned above; and

b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred;

wherein the vehicle is covalently attached to the glucagon antagonist domain. The vehicle and the glucagon antagonist domain may be linked through the N- or C-terminus of the glucagon antagonist domain, as described further below. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain. Preferred glucagon antagonist domains comprise the amino acid sequences described hereinafter in Table 1. Glucagon antagonist domains can be generated by rational design, yeast secretion screening, rational design, protein structural analysis, phage display, RNA-peptide screening and the other techniques mentioned herein.

Further in accordance with the present invention is a process for making therapeutic agents having glucagon antagonist activity, which comprises:

a. selecting at least one peptide having glucagon antagonist activity; and

b. covalently linking said peptide to a vehicle.

The preferred vehicle is an Fc domain. Step (a) is preferably carried out by selection from the peptide sequences in Table 1 hereinafter or from phage display rational design, yeast secretion screening, rational design, protein structural analysis, RNA-peptide screening, or the other techniques mentioned herein.

The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Although non-natural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.

The primary use contemplated for the compounds of this invention is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity that is able to out compete the natural ligand at reasonable therapeutic doses.

The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.

Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary Fc dimers that may be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X¹” and “X²” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:

A, D: Single disulfide-bonded dimers. IgG1 antibodies typically have two disulfide bonds at the hinge region between the constant and variable domains. The Fc domain in FIGS. 1A and 1D may be formed by truncation between the two disulfide bond sites or by substitution of a cysteinyl residue with an unreactive residue (e.g., alanyl). In FIG. 1A, the Fc domain is linked at the amino terminus of the peptides; in 1D, at the carboxyl terminus.

B, E: Doubly disulfide-bonded dimers. This Fc domain may be formed by truncation of the parent antibody to retain both cysteinyl residues in the Fc domain chains or by expression from a construct including a sequence encoding such an Fc domain. In FIG. 1B, the Fc domain is linked at the amino terminus of the peptides; in 1E, at the carboxyl terminus.

C, F: Noncovalent dimers. This Fc domain may be formed by elimination of the cysteinyl residues by either truncation or substitution. One may desire to eliminate the cysteinyl residues to avoid impurities formed by reaction of the cysteinyl residue with cysteinyl residues of other proteins present in the host cell. The noncovalent bonding of the Fc domains is sufficient to hold together the dimer.

Other dimers may be formed by using Fc domains derived from different types of antibodies (e.g., IgG2, IgM).

FIG. 2 shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in this invention.

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

The term “comprising” means that a compound may include additional amino acids on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compound.

The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.

The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.

The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, and R.

The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q.

The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain (which is preferred) as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat. Nos. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published Oct. 28, 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide (e.g., dextran); or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor. Vehicles are further described hereinafter.

The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published Sept. 25, 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference in their entirety. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 1.

The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-inked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR, a succinimnide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴ wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 3 to 75 amino acids, with molecules of 5 to 60 amino acids preferred. Exemplary peptides may comprise known glucagon antagonists, peptides having one or more residues of glucagon randomized, or peptides comprising randomized sequences.

The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods or RNA-peptide screening) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast secretion, RNA-peptide screening, chemical screening, and the like.

The term “glucagon antagonist” refers to a molecule that is able to bind to the glucagon receptor and inhibit the activity of glucagon.

Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. The term “physiologically acceptable salts” refers to any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.

Structure of Compounds

In General. Glucagon binding amino acid sequences are described in Connell (1999), Exp. Opin. Ther. Patents 9(6): 701-709; Unson et al. (1994), J. Biol. Chem. 269(17): 12548-51; Smith et al. (1993), Mol. Pharmacol. 43: 741-8. Each of these references is hereby incorporated by reference in its entirety.

The present inventors identified particular preferred known sequences. These sequences can be randomized through the techniques mentioned above by which one or more amino acids may be changed while maintaining or even improving the binding affinity of the peptide.

In the compositions of matter prepared in accordance with this invention, the peptide may be attached to the vehicle through the peptide's N-terminus or C-terminus. Thus, the vehicle-peptide molecules of this invention may be described by the following formula I:

(A¹)_(a)—F¹—(A²)_(b)  I

wherein:

F¹ is a vehicle (preferably an Fc domain);

A¹ and A² are each independently selected from —(L¹)_(c)—P¹, —(L¹)_(c)—P¹—(L²)_(d)—P², —(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)_(e)—P³, and —(L¹)_(c)—P¹—(L²)_(d)—P²—(L³)_(e)—P³—(L⁴)_(f)—P⁴

P¹, P², P³, and P⁴ are each independently sequences of glucagon antagonist domains;

L¹, L², L³, and L⁴ are each independently linkers; and

a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.

Thus, compound I comprises preferred compounds of the formulae

A¹—F¹  II

and multimers thereof wherein F¹ is an Fc domain and is attached at the C-terminus of A¹;

F¹—A²  III

and multimers thereof wherein F¹ is an Fc domain and is attached at the N-terminus of A²;

F¹—(L¹)_(c)—P¹  IV

and multimers thereof wherein F¹ is an Fc domain and is attached at the N-terminus of —(L¹)_(c)—P¹; and

 F¹—(L¹)_(c)—P¹—(L²)_(d)—P²  V

and multimers thereof wherein F¹ is an Fc domain and is attached at the N-terminus of L¹ P¹ L² P².

Peptides. Any number of peptides may be used in conjunction with the present invention. Peptides may comprise part of the sequence of naturally occurring proteins, may be randomized sequences derived from the sequence of the naturally occurring proteins, or may be wholly randomized sequences. Phage display, yeast-based screening, and RNA-peptide screening, in particular, are useful in generating peptides for use in the present invention.

A glucagon antagonist domain sequence particularly of interest is of the formula

X¹X²X³X⁴X⁵FX⁷X⁸X⁹YX¹¹X¹²X¹³X¹⁴DX¹⁶RRAQX²¹FVQWLMNX²⁹ (SEQ ID NO: 7)

wherein:

X¹ is absent or is an acidic, basic, or hydrophilic residue (D, H, or S preferred);

X² is an amino acid residue (nonfunctional, hydrophilic, or basic residue preferred, A, C, H, P, S, or T most preferred);

X³ is a nonfunctional or hydrophilic residue (Q, L, or M preferred);

X⁴ is an acidic, hydrophilic or nonfunctional residue (A, D, G, or S preferred);

X⁵ is a hydrophilic residue (S or T preferred);

X⁷ is a nonfunctional or hydrophilic residue (I or T preferred);

X⁸ is an acidic or hydrophilic residue (E or S preferred);

X⁹ is an amino acid residue (acidic, nonfunctional, or hydrophilic preferred, A, D, E, L, M, or N most preferred);

X¹¹ is a nonfunctional or hydrophilic residue (A or S preferred);

X¹² is a basic residue (K or R preferred);

X¹³ is a nonfunctional or aromatic residue (A, F, or Y preferred);

X¹⁴ is a nonfunctional or hydrophilic residue (A, L, or N preferred);

X¹⁶ is a nonfunctional or hydrophilic residue (A, Q, or S preferred);

X²¹ is an acidic or nonfunctional residue (D, E, L, or M preferred); and

X²⁹ is an acidic, nonfunctional, or hydrophilic residue (A, E, S, or T preferred).

Exemplary peptide sequences for this invention appear in Table 1 below. Typically, these sequences comprise modifications from the naturally occurring glucagon sequence

His Ser Gin Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr (SEQ ID NO: 8)

Molecules of this invention incorporating the peptide sequences from Table 1 may be prepared by methods known in the art. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well. Any of these peptides may be derivatized as described hereinafter.

TABLE 1 Glucagon Antagonist domains SEQ Description Sequence Reference ID NO: [Glu⁹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Connell 9 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1999), Exp. Gln Trp Leu Met Asn Thr Opin. Ther. Patents 9(6):701-709. [Glu⁹, Arg¹²] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Arg Connell 10 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1999) Gln Trp Leu Met Asn Thr [Glu⁹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Connell 11 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1999) Gln Trp Leu Met Asn Thr [Ala¹¹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Connell 12 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1999) Gln Trp Leu Met Asn Thr [Ala¹⁶] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Connell 13 glucagon Lys Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe (1999) Val Gln Trp Leu Met Asn Thr [Nle⁹, Ala¹¹, His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ala Lys Connell 14 Ala¹⁶] Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1999); Smith glucagon Gln Trp Leu Met Asn Thr et al. (1993), Mol. Pharmacol. 43:741-8. [Nle⁹, Ala¹⁶] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Unson et al. 15 glucagon Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1994), J. Gln Trp Leu Met Asn Thr Biol. Chem. 269(17):1254 8-51. [Ala¹¹ Ala¹⁴] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Unson et al. 16 glucagon Tyr Ala Asp Ser Arg Arg Ala Gln Asp Phe Val (1994) Gln Trp Leu Met Asn Thr [Ala¹¹, Asn¹⁶] His Ser Gln Gly Thr Phe Thr Ser Ala Tyr Ser Lys Unson et al. 17 glucagon Tyr Asn Asp Ser Arg Arg Ala Gln Asp Phe Val (1994) Gln Trp Leu Met Asn Thr [Nle³, Ala¹¹, His Ser Nle Gly Thr Phe Thr Ser Asp Tyr Ala Lys Unson et al. 18 Ala¹⁶] Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Nle³, Ala¹¹, His Ser Nle Gly Thr Phe Thr Ser Asp Tyr Ala Lys Unson et al. 19 Gln¹⁶] Tyr Leu Asp Gln Arg Arg Ala Gln Asp Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Nle⁹ Ala¹⁶] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Unson et al. 20 glucagon Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1994) Gln Trp Leu Met Asn Thr [Ala¹¹ Ala¹⁶] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Unson et al. 21 glucagon Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1994) Gln Trp Leu Met Asn Thr [Ala¹¹ Gln¹⁶] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Unson et al. 22 glucagon Tyr Leu Asp Gln Arg Arg Ala Gln Asp Phe Val (1994) Gln Trp Leu Met Asn Thr [Nle⁹ Ala¹¹ His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ala Lys Unson et al. 23 Ala¹⁶] Tyr Leu Asp Ala Arg Arg Ala Gln Asp Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Nle⁹ Ala¹¹ His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ala Lys Unson et al. 24 Gln¹⁶] Tyr Leu Asp Gln Arg Arg Ala Gln Asp Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Glu⁹ Nle²¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Unson et al. 25 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Nle Phe Val (1994) Gln Trp Leu Met Asn Thr [Nle³ Leu²¹] His Ser Nle Gly Thr Phe Thr Ser Asp Tyr Ser Unson et al. 26 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe (1994) Val Gln Trp Leu Met Asn Thr [Leu³ Leu²¹] His Ser Leu Gly Thr Phe Thr Ser Asp Tyr Ser Unson et al. 27 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe (1994) Val Gln Trp Leu Met Asn Thr [Glu⁹ Nle²¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Unson et al. 28 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Nle Phe Val (1994) Gln Trp Leu Met Asn Thr [Nle⁹ Leu²¹] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Unson et al. 29 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe Val (1994) Gln Trp Leu Met Asn Thr [Glu⁹ Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Unson et al. 30 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe Val (1994) Gln Trp Leu Met Asn Thr [Nle⁹ Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Unson et al. 31 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe Val (1994) Gln Trp Leu Met Asn Thr [Glu⁹ Ala¹¹ His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Unson et al. 32 Ala¹⁶ Glu²¹] Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Glu⁶ Ala¹¹ His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Unson et al. 33 Ala¹⁶ Glu²¹] Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val (1994) glucagon Gln Trp Leu Met Asn Thr [Glu⁹ Nle²¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Smith et al. 34 Tyr Leu Asp Ser Arg Arg Ala Gln Nle Phe Val (1993), Mol. Gln Trp Leu Met Asn Thr Pharmacol 43:741-8. [Nle⁹ Leu²¹] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Smith et al. 35 Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe Val (1993) Gln Trp Leu Met Asn Thr [Leu³ Leu²¹] His Ser Leu Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 36 Lys Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe (1993) Val Gln Trp Leu Met Asn Thr [Nle⁹ Leu²¹] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Smith et al. 37 Tyr Leu Asp Ser Arg Arg Ala Gln Leu Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu⁹ Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Smith et al. 38 Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe Val (1993) Gln Trp Leu Met Asn Thr [Nle⁹ Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Nle Tyr Ser Lys Smith et al. 39 Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu⁹ Ala¹¹ His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Smith et al. 40 Ala¹⁶ Glu²¹] Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu⁶ Ala¹¹ His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Smith et al. 41 Ala¹⁶ Glu²¹] Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu⁹, Ala¹¹] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Smith et al. 42 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu⁹, His²⁴] His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Smith et al. 43 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) His Trp Leu Met Asn Thr [Glu⁹, Phe¹³ His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Smith et al. 44 glucagon Phe Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Asn⁹, Phe¹³] His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Smith et al. 45 glucagon Lys Phe Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Asn⁹, Leu²⁷] His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Smith et al. 46 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Leu Asn Thr [Asn⁹] His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Smith et al. 47 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ala⁹] His Ser Gln Gly Thr Phe Thr Ser Ala Tyr Ser Lys Smith et al. 48 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Ile⁷] His Ser Gln Gly Thr Phe Ile Ser Asp Tyr Ser Lys Smith et al. 49 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Asp¹, Ala², Asp Ala Gln Gly Thr Phe Ile Ser Asp Tyr Ser Lys Smith et al. 50 Ile⁷] Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) glucagon Gln Trp Leu Met Asn Thr [Ala²] His Ala Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Smith et al. 51 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Thr²] His Thr Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 52 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Cys²] His Cys Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 53 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Cys²] His Cys Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 54 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Pro²] His Pro Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 55 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [His³, Ser⁶] His Ser His Gly Thr Ser Thr Ser Asp Tyr Ser Lys Smith et al. 56 glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Ser¹] Ser Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 57 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Asp⁴, Ser⁵] His Ser Gln Asp Ser Phe Thr Ser Asp Tyr Ser Smith et al. 58 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ser⁵] His Ser Gln Gly Ser Phe Thr Ser Asp Tyr Ser Smith et al. 59 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ser⁴] His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Smith et al. 60 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ala⁴] His Ser Gln Ala Thr Phe Thr Ser Asp Tyr Ser Smith et al. 61 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ala⁴] His Ser Gln Ala Thr Phe Thr Ser Asp Tyr Ser Smith et al. 62 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ser⁴, Ala²⁹] His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Smith et al. 63 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Ala [Pro³, Ser²⁹] His Ser Pro Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 64 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Ser [Ser²⁹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 65 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Ser [Glu²¹, Ser²⁹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 66 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe (1993) Val Gln Trp Leu Met Asn Ser [Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 67 glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe (1993) Val Gln Trp Leu Met Asn Thr [Ser⁴] His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Smith et al. 68 Glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Thr [Ala¹¹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Smith et al. 69 Glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr [Glu²¹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 70 Glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Glu Phe (1993) Val Gln Trp Leu Met Asn Thr [Glu²⁹] His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Smith et al. 71 Glucagon Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe (1993) Val Gln Trp Leu Met Asn Glu [Glu⁸] His His Gln Gly Thr Phe Thr Glu Asp Tyr Ser Lys Smith et al. 72 Glucagon Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val (1993) Gln Trp Leu Met Asn Thr

The peptides described in Table 1 preferably are des-His¹ (i.e., His at position 1 is absent), as described in the cited references. For peptides in Table 1 wherein norleucine (Nle) is described in the cited reference, L or M are preferred instead of Nle. Additional useful peptide sequences may result from conservative and/or non-conservative modifications of the amino acid sequences of SEQ ID NO. 7 or of those sequences appearing in Table 1.

Conservative modifications will produce peptides having functional and chemical characteristics similar to those of the peptide from which such modifications are made. In contrast, substantial modifications in the functional and/or chemical characteristics of the peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.

For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alanine scanning mutagenesis).

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-peptide molecules (see preceding formulae) described herein. Exemplary amino acid substitutions are set forth in Table 2.

TABLE 2 Amino Acid Substitutions Original Exemplary Preferred Residues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, 1,4 Diamino- Arg butyric Acid, Gln, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Leu Ala, Norleucine

In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorported by chemical peptide synthesis rather than by synthesis in biological systems.

As noted in the foregoing section “Definition of Terms,” naturally occurring residues may be divided into classes based on common sidechain properties that may be useful for modifications of sequence. For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the peptide that are homologous with non-human orthologs, or into non-homologous regions of the molecule. In addition, one may also make modifications using P or G for the purpose of influencing chain orientation.

In making such modifications, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol. 157: 105-131 (1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to amino acid residues; arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

A skilled artisan will be able to determine suitable variants of the polypeptide as set forth in the foregoing sequences using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a peptide to similar peptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a peptide that are not conserved relative to such similar peptides would be less likely to adversely affect the biological activity and/or structure of the peptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the peptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues that are important for activity or structure in similar peptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the peptides.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a peptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays know to those skilled in the art. Such data could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974); Chou et al. Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou etal. Ann. Rev. Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested (Brenner eta., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl et al., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-8 (1987)), and “evolutionary linkage” (See Home, supra, and Brenner, supra).

Vehicles. This invention requires the presence of at least one vehicle (F¹) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a sidechain.

An Fc domain is the preferred vehicle. The Fc domain may be fused to the N or C termini of the pep tides or at both the N and C termini. Fusion to the N terminus is preferred.

As noted above, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 is one such Fc variant.

3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine).

5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2 (FIG. 4) the leucine at position 15 may be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues.

An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display or RNA-peptide screening or other methods for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).

As noted above, polymer vehicles may also be used for F¹. Various means for attaching chemical moieties useful as vehicles are currently available, see. e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kD to about 50 kD, most preferably from about 5 kD to about 10 kD. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.

Linkers. Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:

(Gly)₃Lys(Gly)₄ (SEQ ID NO: 3);

(Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 4);

(Gly)₃Cys(Gly)₄ (SEQ ID NO: 5); and

GlyProAsnGlyGly (SEQ ID NO: 6).

To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO: 3). Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.

Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above.

Derivatives. The inventors also contemplate derivatizing the peptide and/or vehicle portion of the compounds. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:

1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.

2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.

3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR⁶-wherein R⁶ is lower alkyl].

4. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or benzyloxycarbonyl-NH—(CBZ—NH—), wherein R and R¹ are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.

5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. Exemplary C-terminal derivative groups include, for example, —C(O)R² wherein R² is lower alkoxy or —NR³R⁴ wherein R³ and R⁴ are independently hydrogen or C₁-C₈ alkyl (preferably C₁-C₄ alkyl).

6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.

7. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.

Methods of Making

The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.

Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

The compounds of this invention have pharmacologic activity resulting from their glucagon-antagonist activity. Antagonists of glucagon are useful in treating non-insulin dependent diabetes mellitus (NIDDM).

Pharmaceutical Compositions

In General. The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, buccal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference in their entirety. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Oral dosage forms. Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods”.

The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Pulmonary delivery forms. Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl. 5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumornecrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.

Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.

Buccal delivery forms. Buccal delivery of the inventive compound is also contemplated. Buccal delivery formulations are known in the art for use with peptides.

Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

Specific Preferred Embodiments

The inventors have determined preferred structures for the preferred peptides listed in Table 3 below. The symbol “A” may be any of the linkers described herein or may simply represent a normal peptide bond (i.e., so that no linker is present). Tandem repeats and linkers are shown separated by dashes for clarity.

TABLE 3 Preferred embodiments Peptide SEQ ID Description Molecule Sequence/Structure NO [Gtu⁹ Glu²¹] His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys 73 Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr-Λ-F¹ [Glu⁹ Glu²¹] F¹-Λ- His Ser Gln Gly Thr Glu Thr Ser Asp Tyr 74 Ala Lys Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr [des His¹ Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr 75 Glu⁹ Glu²¹] Leu Asp Ala Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr -Λ-F¹ [des His¹ F¹-Λ- Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala 76 Glu⁹ Glu²¹] Lys Tyr Leu Asp Ala Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr [Glu⁶ Ala¹¹ His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser 77 Ala¹⁶] Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr-Λ-F¹ [Glu⁶ Ala¹¹ F¹-Λ- His Ser Gln Gly Thr Phe Thr Ser Asp Tyr 78 Ala¹⁶] Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr [des His¹ Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys 79 Glu⁶ Ala¹¹ Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Ala¹⁶] Gln Trp Leu Met Asn Thr-Λ-F¹ [des His¹ F¹-Λ- Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser 80 Glu⁶ Ala¹¹ Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Ala¹⁶] Val Gln Trp Leu Met Asn Thr

“F¹” is an Fc domain as defined previously herein. In addition to those listed in Table 3, the inventors further contemplate heterodimers in which each strand of an Fc dimer is linked to a different peptide sequence; for example, wherein each Fc is linked to a different sequence selected from Table 1.

All of the compounds of this invention can be prepared by methods described in PCT appl. no. WO 99/25044.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto, without departing from the spirit and scope of the invention as set forth herein.

80 1 684 DNA Homo sapiens CDS (1)..(684) 1 atg gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg 48 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 96 20 25 30 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 144 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 192 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 240 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 288 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 336 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 384 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 432 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 528 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 576 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 624 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 672 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 tct ccg ggt aaa 684 Ser Pro Gly Lys 225 2 228 PRT Homo sapiens 2 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys 225 3 8 PRT Artificial Sequence Preferred linker 3 Gly Gly Gly Lys Gly Gly Gly Gly 1 5 4 7 PRT Artificial Sequence Preferred linker 4 Gly Gly Gly Asn Gly Ser Gly 1 5 5 8 PRT Artificial Sequence Preferred linker 5 Gly Gly Gly Cys Gly Gly Gly Gly 1 5 6 5 PRT Artificial Sequence Preferred linker 6 Gly Pro Asn Gly Gly 1 5 7 29 PRT Artificial Sequence Glucagon antagonist 7 Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Asp Xaa 1 5 10 15 Arg Arg Ala Gln Xaa Phe Val Gln Trp Leu Met Asn Xaa 20 25 8 29 PRT Artificial Sequence Glucagon antagonist 8 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 9 29 PRT Artificial Sequence Glucagon Antagonist 9 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 10 29 PRT Artificial Sequence Glucagon Antagonist 10 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Arg Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 11 29 PRT Artificial Sequence Glucagon Antagonist 11 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 12 29 PRT Artificial Sequence Glucagon Antagonist 12 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 13 29 PRT Artificial Sequence Glucagon Antagonist 13 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 14 29 PRT Artificial Sequence Glucagon Antagonist 14 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 15 29 PRT Artificial Sequence Glucagon Antagonist 15 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 16 29 PRT Artificial Sequence Glucagon Antagonist 16 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Ala Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 17 29 PRT Artificial Sequence Glucagon Antagonist 17 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Ala Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 18 29 PRT Artificial Sequence Glucagon Antagonist 18 His Ser Xaa Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 19 29 PRT Artificial Sequence Glucagon Antagonist 19 His Ser Xaa Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Gln 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 20 29 PRT Artificial Sequence Glucagon Antagonist 20 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 21 29 PRT Artificial Sequence Glucagon Antagonist 21 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 22 29 PRT Artificial Sequence Glucagon Antagonist 22 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Gln 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 23 29 PRT Artificial Sequence Glucagon Antagonist 23 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 24 29 PRT Artificial Sequence Glucagon Antagonist 24 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ala Lys Tyr Leu Asp Gln 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 25 29 PRT Artificial Sequence Glucagon Antagonist 25 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Xaa Phe Val Gln Trp Leu Met Asn Thr 20 25 26 29 PRT Artificial Sequence Glucagon Antagonist 26 His Ser Xaa Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 27 29 PRT Artificial Sequence Glucagon Antagonist 27 His Ser Leu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 28 29 PRT Artificial Sequence Glucagon Antagonist 28 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Xaa Phe Val Gln Trp Leu Met Asn Thr 20 25 29 29 PRT Artificial Sequence Glucagon Antagonist 29 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 30 29 PRT Artificial Sequence Glucagon Antagonist 30 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 31 29 PRT Artificial Sequence Glucagon Antagonist 31 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 32 29 PRT Artificial Sequence Glucagon Antagonist 32 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 33 29 PRT Artificial Sequence Glucagon Antagonist 33 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 34 29 PRT Artificial Sequence Glucagon Antagonist 34 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Thr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Xaa Phe Val Gln Trp Leu Met Asn Thr 20 25 35 29 PRT Artificial Sequence Glucagon Antagonist 35 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 36 29 PRT Artificial Sequence Glucagon Antagonist 36 His Ser Leu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 37 29 PRT Artificial Sequence Glucagon Antagonist 37 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Leu Phe Val Gln Trp Leu Met Asn Thr 20 25 38 29 PRT Artificial Sequence Glucagon Antagonist 38 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 39 29 PRT Artificial Sequence Glucagon Antagonist 39 His Ser Gln Gly Thr Phe Thr Ser Xaa Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 40 29 PRT Artificial Sequence Glucagon Antagonist 40 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 41 29 PRT Artificial Sequence Glucagon Antagonist 41 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 42 29 PRT Artificial Sequence Glucagon Antagonist 42 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ala Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 43 29 PRT Artificial Sequence Glucagon Antagonist 43 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val His Trp Leu Met Asn Thr 20 25 44 29 PRT Artificial Sequence Glucagon Antagonist 44 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Phe Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 45 29 PRT Artificial Sequence Glucagon Antagonist 45 His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Lys Phe Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 46 29 PRT Artificial Sequence Glucagon Antagonist 46 His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Leu Asn Thr 20 25 47 29 PRT Artificial Sequence Glucagon Antagonist 47 His Ser Gln Gly Thr Phe Thr Ser Asn Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 48 29 PRT Artificial Sequence Glucagon Antagonist 48 His Ser Gln Gly Thr Phe Thr Ser Ala Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 49 29 PRT Artificial Sequence Glucagon Antagonist 49 His Ser Gln Gly Thr Phe Ile Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 50 29 PRT Artificial Sequence Glucagon Antagonist 50 Asp Ala Gln Gly Thr Phe Ile Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 51 29 PRT Artificial Sequence Glucagon Antagonist 51 His Ala Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 52 29 PRT Artificial Sequence Glucagon Antagonist 52 His Thr Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 53 29 PRT Artificial Sequence Glucagon Antagonist 53 His Cys Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 54 29 PRT Artificial Sequence Glucagon Antagonist 54 His Cys Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 55 29 PRT Artificial Sequence Glucagon Antagonist 55 His Pro Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 56 29 PRT Artificial Sequence Glucagon Antagonist 56 His Ser His Gly Thr Ser Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 57 29 PRT Artificial Sequence Glucagon Antagonist 57 Ser Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 58 29 PRT Artificial Sequence Glucagon Antagonist 58 His Ser Gln Asp Ser Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 59 29 PRT Artificial Sequence Glucagon Antagonist 59 His Ser Gln Gly Ser Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 60 29 PRT Artificial Sequence Glucagon Antagonist 60 His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 61 29 PRT Artificial Sequence Glucagon Antagonist 61 His Ser Gln Ala Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 62 29 PRT Artificial Sequence Glucagon Antagonist 62 His Ser Gln Ala Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 63 29 PRT Artificial Sequence Glucagon Antagonist 63 His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Ala 20 25 64 29 PRT Artificial Sequence Glucagon Antagonist 64 His Ser Pro Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Ser 20 25 65 29 PRT Artificial Sequence Glucagon Antagonist 65 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Ser 20 25 66 29 PRT Artificial Sequence Glucagon Antagonist 66 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Ser 20 25 67 29 PRT Artificial Sequence Glucagon Antagonist 67 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 68 29 PRT Artificial Sequence Glucagon Antagonist 68 His Ser Gln Ser Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 69 29 PRT Artificial Sequence Glucagon Antagonist 69 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 70 29 PRT Artificial Sequence Glucagon Antagonist 70 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 71 29 PRT Artificial Sequence Glucagon Antagonist 71 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Glu 20 25 72 29 PRT Artificial Sequence Glucagon Antagonist 72 His Ser Gln Gly Thr Phe Thr Glu Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 73 29 PRT Artificial Sequence Preferred embodiment 73 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 74 29 PRT Artificial Sequence Preferred embodiment 74 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 75 29 PRT Artificial Sequence Preferred embodiment 75 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 76 29 PRT Artificial Sequence Preferred embodiment 76 His Ser Gln Gly Thr Phe Thr Ser Glu Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 77 29 PRT Artificial Sequence Preferred embodiment 77 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 78 29 PRT Artificial Sequence Preferred embodiment 78 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 79 29 PRT Artificial Sequence Preferred embodiment 79 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 80 29 PRT Artificial Sequence Preferred embodiment 80 His Ser Gln Gly Thr Glu Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 

What is claimed is:
 1. A composition of matter of the formula (A¹)_(a)—F¹-(A²)_(b) and multimers thereof, wherein: F¹ is a vehicle; A¹ and A² are each independently -(L¹)_(c)-P¹; each P¹ is a randomized glucagon antagonist domain sequence; each L¹ is a linker; and a, b, and c are each independently 0 or 1, provided that at east one of a and b is
 1. 2. The composition of matter of claim 1 of the formulae A¹-F¹ or F¹-A².
 3. The composition of matter of claim 1 of the formula F¹-(L¹)_(c)-P¹.
 4. The composition of matter of claim 1, wherein F¹ is an Fc domain.
 5. The composition of matter of claim 1 wherein F¹ is an IgG Fc domain.
 6. The composition of matter of claim 1 wherein F¹ is an IgG1 Fc domain.
 7. The composition of matter of claim 1 wherein F¹ comprises the sequence of SEQ ID NO:
 2. 8. The composition of matter of claim 1 wherein the glucagon antagonist domain sequence comprises the formula (SEQ ID NO: 7) X¹X²X³X⁴X⁵FX⁷X⁸X⁹X¹¹X¹²X¹³X¹⁴DX¹⁶RRAQX²¹FVQWLMNX²⁹

wherein: X¹ is absent or is an acidic, basic, or hydrophilic residue; X² is an amino acid residue; X³ is a nonfunctional or hydrophilic residue; X⁴ is an acidic, hydrophilic or nonfunctional residue; X⁵ is a hydrophilic residue; X⁷ is a nonfunctional or hydrophilic residue; X⁸ is an acidic or hydrophilic residue; X⁹ is an amino acid residue; X¹¹ is a nonfunctional or hydrophilic residue; X¹² is a basic residue; X¹³ is a nonfunctional or aromatic residue; X¹⁴ is a nonfunctional or hydrophilic residue; X¹⁶ is a nonfunctional or hydrophilic residue; X²¹ is an acidic or nonfunctional residue; and X²⁹ is an acidic, nonfunctional, or hydrophilic residue.
 9. The composition of matter of claim 8, wherein F¹ is an Fc domain.
 10. The composition of matter of claim 8, wherein F¹ is an IgG Fc domain.
 11. The composition of matter of claim 10, wherein F¹ is an IgG1 Fc domain.
 12. The composition of matter of claim 8, wherein: X¹ is absent or is H, D or S; X² is A, C, H, P, S, or T; X³ is L, M, or Q; X⁴ is A, D, G, or S; X⁵ is S or T; X⁷ is I or T; X⁸ is E or S; X⁹ is A, D, E, L, M, or N; X¹¹ is A or S; X¹² is K or R; X¹³ is A, F, or Y; X¹⁴ is A, L, or N; X¹⁶ is A, Q, or S; X²¹ is D, E, L, or M; X²⁹ is A, E, S, or T.
 13. The composition of matter of claim 1, wherein the glucagon antagonist sequence is selected from Table 1 (SEQ ID NOS: 9 to 72).
 14. The composition of matter of claim 8, wherein the glucagon antagonist sequence is selected from Table 1 (SEQ ID NOS: 9 to 72).
 15. The compound of claim 4, wherein the C-terminus is amidated. 